FIGURE 3:

Analysis of endogenous septins and hetero-oligomerization in cell lines expressing AcGFP-tagged SEPT9 isoform reporters. K562 cells were transfected with pMEP-based, episomally replicating vectors directing regulatable expression of the indicated SEPT9 derivative. Cells were counterselected with hygromycin, and expression was controlled as outlined in Materials and Methods. (A) Western blot detection of septins expressed by K562 cells. Septins belonging to the SEPT2, SEPT3, and SEPT6 homology-based subgroups are indicated at the left margin. (B) Crude extract of cells transfected with the indicated AcGFP-SEPT9 isoform was analyzed by density-gradient centrifugation. Transfection conditions were adjusted for modest expression levels, which correspond to ∼50% of the level in A. The distribution of heteromers (detection based on SEPT7) and AcGFP-SEPT9 was analyzed by Western blotting. Note that six- and eight-subunit heteromers are not resolved by this method and that a 26.9-kDa AcGFP-fusion partner is too small to detectably alter sedimentation profiles. Sedimentation peaks for proteins of known S values are indicated by vertical dotted lines.